Roth S, Osinski J V, Park S S, ostwald P, Moshfeghi A A
Department of Anesthesia and Critical Care, University of Chicago, IL 60637, USA.
J Neurosci Methods. 1996 Sep;68(1):87-90. doi: 10.1016/0165-0270(96)00061-1.
Adenosine, produced from the decomposition of adenosine triphosphate, is believed to provide protective effects during ischemia. On the other hand, adenosine metabolites may serve as precursors for oxygen free radical formation. These substances have not been previously measured in intact vertebrate retina, where adenosine and its metabolites may play a role in the pathogenesis of ischemic injury. The small tissue mass of the retina, particularly in rats, renders these measurements challenging. Furthermore, accurate measurement of purine nucleosides requires immediate cessation of ongoing adenosine metabolism. Concentrations of adenosine and its purine nucleoside metabolites inosine, hypoxanthine, and xanthine in the retina of ketamine/xylazine-anesthetized rats were measured after in situ freezing using high-performance liquid chromatography. The retina was removed from the frozen eyes and analyzed. Quantitative measurements were made possible through the use of an internal standard. Ischemia was induced by ligation of the central retinal artery. Retinal purine nucleoside concentrations did not differ between the two eyes of the rat under control conditions, and there was no effect of placement of the ligating suture itself compared to completely unmanipulated eyes. Use of two different in situ freezing methods yielded comparable results. To evaluate the impact of a period of ischemia, one retina of each rat was ischemic for 30 min, and the other, non-ischemic. Our measurements were associated with a high degree of reproducibility and minimal variability, and significant changes in purine nucleoside concentrations were detectable in the retina after 30 min of ischemia. Our method may be used to assess the role of adenosine and its metabolites in the pathogenesis of ischemic neuronal injury, including in the retina.
由三磷酸腺苷分解产生的腺苷被认为在缺血期间具有保护作用。另一方面，腺苷代谢产物可能是氧自由基形成的前体。这些物质此前尚未在完整的脊椎动物视网膜中进行测量，而腺苷及其代谢产物可能在缺血性损伤的发病机制中发挥作用。视网膜的组织质量较小，尤其是在大鼠中，这使得这些测量具有挑战性。此外，准确测量嘌呤核苷需要立即停止正在进行的腺苷代谢。使用高效液相色谱法在原位冷冻后，测量了氯胺酮/赛拉嗪麻醉大鼠视网膜中腺苷及其嘌呤核苷代谢产物肌苷、次黄嘌呤和黄嘌呤的浓度。将视网膜从冷冻的眼睛中取出并进行分析。通过使用内标实现了定量测量。通过结扎视网膜中央动脉诱导缺血。在对照条件下，大鼠的两只眼睛之间视网膜嘌呤核苷浓度没有差异，与完全未处理的眼睛相比，结扎缝线的放置本身没有影响。使用两种不同的原位冷冻方法得到了可比的结果。为了评估一段缺血时间的影响，每只大鼠的一个视网膜缺血30分钟，另一个视网膜不缺血。我们的测量具有高度的可重复性和最小的变异性，并且在缺血30分钟后视网膜中嘌呤核苷浓度有明显变化。我们的方法可用于评估腺苷及其代谢产物在缺血性神经元损伤发病机制中的作用，包括在视网膜中的作用。
